The goal of our proposed research is to provide a detailed structural description of how O2 binding to one subunit of the hemoglobin (Hb) molecule can alter the accessibility and reactivity of the heme-iron atoms in adjacent subunits. Even though Hb is one of the best-studied proteins, many details of its structure and function are not fully understood and several aspects are controversial. Simple two-structure allosteric models cannot account for the stereochemical mechanism for the cooperative oxygenation of Hb. For example, there are at least three quaternary structures (T, R, and R2) of Hb in crystals and the functional properties of Hb in crystals are distinctly different from those in solution. Recent multinuclear nuclear magnetic resonance (NMR) results indicate that the solution structure of human normal adult Hb in the CO form is a dynamic ensemble of the R and R2 crystal structures. In order to correlate the structure, dynamics, and function of Hb under physiological conditions and to resolve the conflicting results derived from studies obtained from Hb crystals and Hb in solution, we need to know the details of the structures and dynamics of Hb as a function of oxygenation in solution. We plan to apply techniques of biochemistry, biophysics, molecular biology, and structural biology to our Hb research and to correlate the results obtained from NMR spectroscopy, X-ray crystallography, resonance Raman spectroscopy, computer modeling, and equilibrium and kinetic studies of the binding of O2 and other ligands to Hb. With our Escherichia coli expression system for Hb, we can express any desired mutants of Hb that can aid in our study. Our specific aims are: (i) to determine the relative functional and structural importance of changes at the a^ interface vs the a$2 interface;(ii) to evaluate the effects of differential ligand binding to the a- and (3-chains on the expression of cooperative O2 binding;and (iii) to investigate the effects of allosteric effectors (H+ ions and organic phosphates) on the proximal geometry and distal accessibility of the heme-iron atoms to describe the structural basis behind the Bohr effect and the effect of organic phosphates. The knowledge that we gain can also provide insights into the development of a new generation of Hb-based oxygen carriers and of a new approach to the treatment of patients with sickle cell anemia. Hb research is a good illustration of how discoveries from basic research on proteins can make important contributions to medicine and technology.